The erbB/EGF receptor related gene family encodes growth factor receptors with intrinsic tyrosine kinase activity. Four members of this family have been identified: ErbB/EGF receptor, ErbB-2, ErbB-3 and ErbB4 (reviewed in Peles and Yarden, 1993). Members of this family have been implicated in the development of a variety of human malignancies. EGF receptor gene amplification and overexpression has been observed in a high percentage of primary human carcinomas of epithelial origin including glioblastoma, cancer of the lung, breast, head and neck, and bladder, and correlates with an unfavorable prognosis for the patients (reviewed in Gullick, 1991). Increased receptor expression in tumor cells is often accompanied by increased production of TGF.alpha. (Derynck et al., 1987), which leads to receptor activation by an autocrine pathway and contributes to malignant transformation. Due to its accessibility on the cell surface, its overexpression in several types of cancer, and its involvement as a marker for an unfavorable prognosis, the EGF receptor and/or its variants (Ekstrand, A. J., Longo, N., Hamid, M. L., Olson, J. J., Liu, L., Collins, V. P., and James, C. D. Functional characterization of an EGF receptor with a truncated extracellular domain expressed in glioblastomas with EGFR gene amplification. Oncogene, 9: 2313-2320, 1994; Sugawa, N., Ekstrand, A. J., James, C. D., and Collins, V. P. Identical splicing of aberrant epidermal growth factor receptor transcripts from amplified rearranged genes in human glioblastomas. Proc. Natl. Acad. Sci. U.S.A., 87: 8602-6, 1990; Wong, A. J., Ruppert, J. M., Bigner, S. H., Grzeschik, C. H., Humphrey, P. A., Bigner, D. S., and Vogelstein, B. Structural alterations of the epidermal growth factor receptor gene in human gliomas. Proc. Natl. Acad. Sci. U.S.A., 89: 2965-9, 1992; Yamazaki, H., Ohba, Y., Tamaoki, N., and Shibuya, M. A deletion mutation within the ligand binding domain is responsible for activation of epidermal growth factor receptor gene in human brain tumors. Jpn. J. Cancer Res., 81: 773-9, 1990; Wikstrand, C. J., Hale, L. P., Batra, S. K., Hill, M. L., Humphrey, P. A., Kurpad, S. N., McLendon, R. E., Moscatello, D., Pegram, C. N., Reist, C. J., and et al Monoclonal antibodies against EGFRvIII are tumor specific and react with breast and lung carcinomas and malignant gliomas. Cancer Res., 55: 3140-3148, 1995; Moscatello, D. K., Holgado-Madruga, M., Godwin, A. K., Ramirez, G., Gunn, G., Zoltick, P. W., Biegel, J. A., Hayes, R L., and Wong, A. J. Frequent expression of a mutant epidermal growth factor receptor in multiple human tumors. Cancer Res., 55: 5536-9, 1995; Hills, D., Rowlinson-Busza, G., and Gullick, W. J. Specific targeting of a mutant, activated EGF receptor found in glioblastoma using a monoclonal antibody. Int. J. Cancer, 63: 537-543, 1995) are under intensive scrutiny as a therapeutic target for novel antitumor reagents.
Various strategies have been employed to target the EGF receptor and/or his variants for tumor therapy. Monoclonal antibodies directed towards the extracellular domain of the EGF receptor have proven effective in the inhibition of tumor cell growth. The EGF receptor specific Mab 225 competes with EGF for binding to the EGF receptor thereby blocking ligand dependent receptor activation (Fan et al., 1993). Treatment with Mab 225 inhibits the growth of EGF receptor expressing tumor cells in vitro and in animal models in vivo (Masui et al., 1984; Ennis et al., 1989), and clinical studies with Mab 225 have recently been initiated. In an attempt to achieve more potent antitumoral effects recombinant fusion proteins have been constructed which contain the enzymatic domains of Pseudomonas exotoxin A (Chaudhary et al., 1987) or diphtheria toxin (Shaw et al., 1991), and employ the natural EGF receptor ligands TGF.alpha. or EGF for targeting to receptor overexpressing tumor cells. Due to the growth factor domain such toxins are able to activate the EGF receptor (Schmidt and Wels, 1996), which might facilitate rapid uptake by tumor cells, but could also be responsible for the significant cytotoxic activity displayed on cells expressing only moderate levels of the target receptor.
As an alternative to growth factors, single-chain immunotoxin (SCIT) fusion proteins can be used for the target cell specific delivery of therapeutic effector functions. SCIT fusion proteins are hybrid molecules that contain antibody variable regions (scFv) genetically fused to a binding defective toxin, most notably diphteria toxin (DT) and Pseudomonas exotoxin A (ETA). Both DT and PE halt protein synthesis by ADP-ribosylating elongation factor 2 upon entry to the cell cytosol, resulting in cell death (Carroll and Collier, 1987). A recombinant single chain antibody-toxin consisting of a scFv domain derived from the antagonistic Mab 225 and truncated Pseudomonas exotoxin A was described recently (Wels et al., 1995).